Type 1 diabetes is a Thl lymphocyte mediated autoimmune disease that is partially suppressed by oral administration of small amounts of insulin or other diabetes autoantigens. To strengthen this promising immunotolerization approach to diabetes prevention, new innovative strategies are needed. In response to RFA-DK-02-023, we propose to determine the feasibility of a recombinant vaccinia virus (rVV) mediated autoantigen delivery system for suppression of Type 1 diabetes. Genes encoding proinsulin (INS) and glutamate decarboxylase (GAD) autoantigens fused to a cholera toxin B subunit (CTB) receptor IJgand in an rVV expression vector will be expressed in intestinal epithelial cells inoculated with rVV. Following enterocyte apoptosis, autoantigen fusion proteins will be taken up by APCs of the subepithelium or released into the gut and targeted via the CTB ligand to intestinal M cells for indirect APC processing. Two specific aims will determine (1) rVV MOl's required to generate optimum autoantigen protein synthesis in enterocyte cells in culture. (2) The efficacy of rVV mediated tolerance for prevention of Type1 diabetes in vivo and the nature of the T cell response underlying diabetes prevention. Prediabetic NOD/LtJ mice will be orally inoculated with rVV encoding CTB-INS and CTB-GAD enterocyte targeted autoantigen fusion proteins to generate maximum levels of protection from diabetes as measured by levels of insulitis and hyperglycemia. Mechanisms of immunity underlying vaccinia mediated autoantigen suppression of diabetes will be investigated by measurement of T lymphocyte responses to mucosal immunization measured by ELISA quantification of IL-10, TGF-beta, IL-2 and IFN-gamma cytokines secreted from salivary gland, spleen, pancreas and mesenteric lymphoid cells. CTL and T lymphocyte proliferation in response to vaccina mediated synthesis of INS and GAD will be quantified by flow cytometric analysis of spleen and mesenteric lymph node memory CD4+ and CD8+ lymphocytes. Specific CTL responses to vaccinia infection will be quantified by LDH assays for rVV, CTB, INS and GAD proteins. The results of these experiments will increase our understanding of the immunological mechanisms underlying vaccinia virus mediated immunotolerization against Type 1 diabetes for the long term goal of generating safer, more effective and inexpensive mucosal vaccines for protection against this devastating form of diabetes.